Immunological detection methods

ABSTRACT

The present invention relates to monoclonal antibodies that are distinguished by a high degree of selectivity and affinity towards triasulfurone and that are therefore outstandingly suitable for use in an immunoassay for the rapid and effective detection of triasulfurone. The present invention relates also to hybridoma cell lines that produce the said monoclonal antibodies and to immunological methods for the detection of triasulfurone in soil, water or air samples using the said monoclonal antibodies and to test kits that may be used in those detection methods.

This is a divisional of Ser. No. 08/159,030, filed Nov. 29, 1993, nowU.S. Pat. No. 5,359,135, which is a divisional of Ser. No. 07/722,650,filed Jun. 28, 1991, now abandoned.

The present invention relates to monoclonal antibodies that aredistinguished by a high degree of selectivity and affinity towardsherbicides from the group of the sulfonylureas, for exampletriasulfurone, and that are therefore outstandingly suitable for use inan immunoassay for the rapid and effective detection of sulfonylureaherbicides in soil, water or air samples, or in plant extracts, and tomethods for the preparation of said monoclonal antibodies.

The present invention relates also to hybridoma cell lines that producesaid monoclonal antibodies and to immunological methods for thedetection of sulfonylurea herbicides in soil, water or air samples andin biological material, for example plant extracts, using saidmonoclonal antibodies, and to the test kits that may be used in thosedetection methods.

Recently, the use of synthetic herbicides for plant protection purposesand the environmental problems associated therewith have beenincreasingly at the forefront of public discussion.

Sulfonylureas are a new class of especially effective herbicides thatare used for plant protection purposes, especially for the selective andeffective control of broad-leaved weeds in cereal crops.

A representative of that class is triasulfurone[3-(6-methoxy-4-methyl-1,3,5-triazin-2-yl)-1-[2-(2-chloroethoxy)-phenylsulfonyl]-urea],which is used especially in small-grained cereal crops for the selectivecontrol of broad-leaved weeds, for example Viola tricolor or Galiumaparine.

The detection of herbicides from the group of the sulfonylureas in soiland water samples is at present carded out mainly by means of gas orliquid chromatography (for example HPLC) [GC: Ahmad and Crawford (1990);HPLC: Zahnow (1982); Iwanzik and Egli (1988); van Rensburg E (1985)] andusing bioassays.

Since in some cases even very small residues of those herbicides in thesoil still exhibit a high degree of residual activity, which may beharmful especially to subsequent crops that are susceptible tosulfonylureas, it is of great importance to have available a reliableand extremely sensitive detection method for residue analysis.

Although most of the methods mentioned above for the detection ofsynthetic herbicides do have the sensitivity necessary for residueanalysis, their use is generally associated with a large number ofdisadvantages. For example, for the determination of triasulfurone insoil samples using GC or HPLC, laborious purifying and concentratingsteps have to be effected before the actual chromatographic analysis iscarried out.

Other disadvantages of those methods are, for example, that, in gaschromatography, detectors that are element-specific only are used, whilein the case of HPLC photometric detectors, which are relativelynon-specific, are used. With the exception of detection by massspectroscopy, the chromatographic analyses rely on the determination ofthe retention times for the particular substance. Those values are,however, relative and are therefore not structure-specific.

The bioassay methods that are likewise mentioned above can be carriedout without a great deal of effort, but are not sufficiently specific.

In order to avoid the above-described disadvantages of the establishedanalytical methods, attempts have recently been made to developimmunological methods--such as are already used routinely in clinicaldiagnosis for the detection of a wide variety of antigens--for theagricultural sector also, especially for the quantitative andqualitative determination of agricultural chemicals in soil, water orair samples.

For example, the development of immunological methods for the detectionof specific herbicides, such as 2,4-dichlorophenoxyacetic acid (Fleeker,1986) or chlorosulfurone (Kelley et al, 1985), and of variouspesticides, such as difluobenzurone (Wie and Hammock, 1982), metalaxyl(Newsome, 1985), alachlor (Feng et al, 1990) or parathion (Ercegovich etal, 1981) is already underway.

Methods for the immunological detection of herbicides from the group ofthe sulfonylureas have thus already been described [Kelly et al (1985)],but they, like the methods described hereinbefore, rely on the use ofpolyclonal antisera obtained from animals that have previously beenimmunised with a corresponding antigen. The preparation of monoclonalantibodies to sulfonylurea herbicides that have a sufficiently highdegree of affinity towards the target substance and are thereforesuitable for use according to the invention in one of the knownimmunoassays has not yet been successful, however.

Polyclonal antisera are of very heterogeneous composition, that is tosay they comprise a large number of different antibodies that react withdifferent epitopes of the particular antigen. This heterogeneouscomposition of polyclonal antisera results from the fact that wheneveran experimental animal is immunised with a specific antigen, severalantibody-producing cell clones are stimulated at the same time; each ofthose clones recognises a different epitope on the antigen molecule and,as a result, different antibodies are produced by the stimulated cellclones.

For that reason, sera of immunised animals are always polyclonal andtherefore heterogeneous, as regards both their specificity and theirmembership of the individual classes of immunoglobulins.

This heterogeneity of the composition of polyclonal antisera maytherefore lead to a situation in which, owing to insufficient affinity,their detection sensitivity is not adequate for the selective detectionof a specific target substance, or in which structurally closely relatedcompounds, for example triasulfurone and its hydroxylation products,cannot be adequately differentiated when using polyclonal antibodies inthe context of an immunoassay.

In order to overcome those disadvantages of the polyclonal antisera,efforts aimed at the development of monoclonal antibodies for theagricultural sector also have recently increased. For example Schlaeppiet al (1989) report on the preparation and use of monoclonal antibodiesto atrazine and hydroxyatrazine in an enzyme-linked immunoassay.

The problem to be solved within the context of this invention was thusespecially to provide an immunoassay for the rapid and reliabledetection of sulfonylurea herbicides, especially of triasulfurone, insoil, water and air samples that was easy to use, effective and highlyselective.

This problem has now been solved, surprisingly, within the context ofthe present invention by providing monoclonal antibodies having a highdegree of specificity and affinity towards sulfonylurea herbicides,especially towards triasulfurone, using the hybridoma/monoclonalantibody technology that is known per se.

The use of hybrid somatic cell lines (hybridomas) as the source forantibodies to quite specific antigens dates back to the work of K ohlerand Milstein (Nature, 256: 495-97, 1975).

The antibodies that can be obtained by means of the method describedtherein differ very greatly from those that are obtained from antiseraof conventionally immunised animals.

The principle of hybridoma/monoclonal antibody technology is based onthe observation that when two somatic cells fuse, the resulting hybridcell exhibits characteristic features of both parent types.

In the case of monoclonal antibody production, the ability to synthesisea specific antibody originates from an immunocompetent B cell (usually aspleen cell) taken from a previously immunised donor animal, while thecapacity for continuous cell division in culture is supplied by theother fusion partner, a tumour cell line (often a myeloma).

The donor animals are generally immunised using conjugates consisting ofthe target molecule and a high molecular weight carrier molecule linkedthereto.

Each of those hybrid cell lines synthesises a homogeneous immunoglobulinthat is only one single representative from the large number of possibleantibodies that can be synthesised in vivo by an animal in response toan antigen.

Since each immunoglobulin-producing clone is characterised by a singletype of antibody, the expression "monoclonal antibody" has becomeaccepted.

The advantages of monoclonal antibodies over polyclonal antibodies aremany:

a) monoclonal antibodies can be obtained in large numbers and in a highdegree of purity,

b) the preparation of monoclonal antibodies is homogeneous as regardsthe antigen reactivity and does not change over time,

c) monoclonal-antibody-producing hybridomas can be stored for years anddecades without losing their specific properties, i.e. the production ofspecific monoclonal antibodies,

d) monoclonal antibodies are better suited for use as standard reagentsthan are polyclonal antisera, since the latter are adversely affected bya large spectrum of variation, for example as regards

α) the removal of blood from immunised animals for the purpose ofobtaining antiserum.

β) constant availability of material for additional immunisation,

γ) the limited lifespan of the donor animals.

Monoclonal antibodies, which have now been produced to a large number ofantigens, are well established especially in medical diagnosis, wheretheir use is now quite indispensable.

Within the context of the present invention, there has now been madeavailable for the first time a method that, using thehybridoma/monoclonal antibody technology that is known per se and isoutlined briefly above, allows the production of monoclonal antibodieshaving a high degree of specificity and affinity towards sulfonylureaherbicides, especially towards triasulfurone, that are suitable byvirtue of their high degree of affinity for use according to theinvention in one of the known immunoassays.

In particular, the method is characterised essentially by the fact thatin the preparation of conjugates for the immunisation of the donoranimals there is used instead of the whole sulfonylurea moleculeperferably only a fragment that preferably includes the sulfonamidemoiety of the molecule, and that that fragment is linked to one of thehigh molecular weight carrier molecules customarily used.

After the immune response has been triggered in the donor animal, thecells responsible for the production of antibodies are isolated from thedonor animal, and fused with suitable myeloma cells in order to producehybridoma cells, in the manner described in detail below.

The present invention thus particularly relates to a method for theproduction of a monoclonal antibody having a high degree of specificityand affinity towards one or more sulfonylurea herbicides, wherein

(a) a linking component comprising essentially the sulfonamide moiety ofthe target molecule is conjugated with a suitable high molecular weightcarrier molecule;

(b) a donor animal is immunised with the conjugate prepared inaccordance with (a);

(c) immunocompetent B cells are isolated from the immunised donoranimal;

(d) the said immunocompetent B cells are fused with tumour cells capableof continuous cell division;

(e) the resulting fusion product is isolated and after selection thehybridoma cells that produce the desired antibody are cloned, and

(f) the said hybridoma cells are cultured in vitro or in vivo to producemonoclonal antibodies.

The present invention relates also to the monoclonal antibodiesresulting from the method according to the invention. Preferred aremonoclonal antibodies having a high degree of specificity and affinitytowards sulfonylurea herbicides, especially towards triasulfurone, thatare outstandingly suitable by virtue of their low cross-reactivity foruse in an immunoassay for the rapid and reliable detection ofsulfonylurea herbicides, especially of triasulfurone, and that cantherefore be used also for differentiating the target substances, suchas triasulfurone, from structurally related compounds, especially fromthe hydroxylation products and from inactive metabolites.

Special preference is therefore given within the context of the presentinvention to monoclonal antibodies and derivatives thereof that have ahigh degree of specificity and affinity towards triasulfurone and thatexhibit essentially no cross-reactivity with a large number oftriasulfurone analogs, especially triasulfurone analogs selected fromthe group consisting of primisulfurone, sulfometurone-methyl,tribenurone-methyl, thiameturone-methyl, chlorosulfurone,bensulfurone-methyl, metsulfurone-methyl, nicosulfurone-methyl and DPX9636.

Special preference is likewise given to monoclonal antibodies that havea high degree of specificity and affinity towards triasulfurone and thatexhibit a cross-reactivity of <1.0%, especially of <0.7%, withstructurally related triasulfurone analogs selected from the groupconsisting of primisulfurone, sulfometurone-methyl, tribenurone-methyl,thiameturone-methyl, chlorosulfurone, bensulfurone-methyl,metsulfurone-methyl, nicosulfurone-methyl and DPX 9636.

Especially preferred are monoclonal antibodies obtainable from hybridomacell lines that have the distinguishing characteristics of ECACC 90021702 or ECACC 9002 1703, as well as derivatives of said monoclonalantibodies.

Likewise especially preferred are monoclonal antibodies that have a highdegree of specificity and affinity towards triasulfurone and thatexhibit a cross-reactivity of <0.1%, especially of ≦0.01%, withstructurally related triasulfurone analogs selected from the groupconsisting of primisulfurone, sulfometurone-methyl, tribenurone-methyl,thiameturone-methyl, chlorosulfurone, bensulfurone-methyl,metsulfurone-methyl, nicosulfurone-methyl and DPX 9636.

Very especially preferred is a monoclonal antibody obtainable from ahybridoma cell line that has the distinguishing characteristics of ECACC9002 1704, and derivatives of said monoclonal antibody.

Within the context of the present invention, derivatives of monoclonalantibodies are to be understood as being, for example, antibodyfragments that still have the high degree of specificity and affinityfor the antigenic determinants of triasulfurone, as well asradioactively labelled monoclonal antibodies that are labelled, forexample, with radioactive iodine (¹²⁵ I, ¹³¹ I), carbon (¹⁴ C), sulfur(³⁵ S), tritium (³ H) or the like, conjugates of monoclonal antibodieswith biotin or avidin, with enzymes, such as horseradish peroxidase,alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase,carboxylic acid anhydrase, acetylcholine esterase, lysozyme, malatedehydrogenase or glucose 6-phosphate dehydrogenase, also conjugates ofmonoclonal antibodies with bioluminescent agents (for exampleluciferase), chemoluminescent agents (for example acridine esters) orfluorescent agents (for example phycobiliproteins). The presentApplication likewise includes bispecific and cross-linked antibodies.

This list of examples of possible antibody derivatives serves merely toillustrate the present invention and is not intended to limit thesubject of the invention in any way.

Within the context of this invention, the expression "essentially nocross-reactivity" is intended to be understood as meaning that thereactivity of the monoclonal antibodies specific to triasulfurone withnon-specific epitopes of other compounds, especially structurallyrelated compounds, such as the hydroxylation products, is in themajority of cases less than 10%, and preferably less than 1.5%,especially less than 0.1%.

As defined within the context of this invention, the percentage ofcross-reactivity is represented by the following equation:

    [triasulfurone concentration for 50% inhibition (I.sub.50)/concentration of the triasulfurone analogs for 50% inhibition (I.sub.50)]×100.

The I₅₀ value can be determined, for example, by means of a competitiveELISA assay (see Example 8). In that case the I₅₀ value corresponds, forexample, to the antigen concentration that leads to a 50% inhibition ofthe binding of the antibody to the carrier-bound antigen.

The present invention relates further to hybridoma cell lines thatsynthesise and preferably secrete into the surrounding medium themonoclonal antibodies characterised in detail hereinbefore.

The present invention relates especially to a hybridoma cell line thatproduces a monoclonal antibody that has a high degree of specificity andaffinity towards triasulfurone and that exhibits essentially nocross-reactivity with a large number of structually related compounds,especially with those selected from the group consisting ofprimisulfurone, sulfometurone-methyl, tribenurone-methyl,thiameturone-methyl, chlorosulfurone, bensulfurone-methyl,metsulfurone-methyl, nicosulfurone-methyl and DPX 9636.

Preference is given to a hybridoma cell line that synthesises andpreferably secretes into the surrounding medium a monoclonal antibodythat has a high degree of specificity and affinity towards triasulfuroneand that exhibits a cross-reactivity of <10%, especially of <1.5%, andmost especially of <0.1%, with a large number of structurally relatedtriasulfurone analogs.

Special preference is given to a hybridoma cell line that synthesisesand preferably secretes into the surrounding medium a monoclonalantibody that has a high degree of specificity and affinity towardstriasulfurone and that exhibits a cross-reactivity of <1.0%, especiallyof <0.7%, with structurally related triasulfurone analogs selected fromthe group consisting of primisulfurone, sulfometurone-methyl,tribenurone-methyl, thiameturone-methyl, chlorosulfurone,bensulfurone-methyl, metsulfurone-methyl, nicosulfurone-methyl and DPX9636.

Very special preference is given to hybridoma cell lines that have thedistinguishing characteristics of ECACC 9002 1702 or ECACC 9002 1703,and to the clones and sub-clones thereof.

Special preference is given also to a hybridoma cell line thatsynthesises and preferably secretes into the surrounding medium amonoclonal antibody that has a high degree of specificity and affinitytowards triasulfurone and that exhibits a cross-reactivity of <0.1%,especially of ≦0.01%, with structurally related triasulfurone analogsselected from the group consisting of primisulfurone,sulfometurone-methyl, tribenurone-methyl, thiameturone-methyl,chlorosulfurone, bensulfurone-methyl, metsulfurone-methyl,nicosulfurone-methyl and DPX 9636.

Very special preference is given to a hybridoma cell line that has thedistinguishing characteristics of ECACC 9002 1704, and to the clones andsub-clones thereof.

The present invention relates also to variants and mutants of thehybridoma cell lines characterised in detail above that occurspontaneously or that can be produced artificially using known methodsand that still have the characteristic properties of the startingmaterial, that is to say are still capable of producing the antibodiesaccording to the invention or derivatives thereof and preferably ofsecreting them into the surrounding medium.

The present invention also includes methods for the production of thesaid hybridoma cell lines and to methods for the production of the saidmonoclonal antibodies.

Clones and sub-clones of hybridoma cell lines are to be understood asbeing hybridomas that are produced from the starting clone by repeatedcloning and that still have the features essential to the invention ofthe starting clone.

The invention relates also to a method for the immunological detectionof sulfonylurea herbicides, especially of triasulfurone, for example insoil, water or air samples and in biological material, for example inplant or animal extracts, using the monoclonal antibodies according tothe invention.

The present invention also includes means for the qualitative andquantitative determination of sulfonylurea herbicides, especially oftriasulfurone, in the form of ready-to-use test kits that comprise atleast one of the monoclonal antibodies according to the invention asreagent and that are suitable for use under field conditions for therapid and reliable detection of sulfonylurea herbicides, especially oftriasulfurone.

The monoclonal antibodies according to the invention are produced usingmethods known per se that are based essentially on the methods developedby K ohler und Milstein (1975).

Since the target substances [sulfonylurea herbicides] to be analysed,for example triasulfurone, for which specific monoclonal antibodies areto be developed are relatively small and simple molecules which afteradministration to an experimental animal are not capable alone oftriggering a corresponding immune response in that animal, preparatorymeasures have to be taken before the actual immunisation.

Compounds that owing to their size and simple structure are not capableof inducing an immune reaction are described as haptens or incompleteantigens and are therefore distinguished from the complete antigens(=immunogens) which both act as antigens and are capable of inducing animmune response. Such hapten molecules may be conjugated to highmolecular weight compounds (carrier molecules), as a result of whichthey become comparable to complete antigens as regards theircharacteristics, that is to say they are then capable of triggering animmune response.

Some of the antibodies formed in the course of the immunisation reactionare then capable of reacting with specific epitopes on the haptenmolecule, regardless of whether the hapten molecule is present alone oris still linked to the carrier molecule.

The term hapten, which is used frequently hereinbelow, is to beunderstood within the context of this invention as meaning primarily thetriasulfurone molecule or a part thereof used for the immunisation.

Within the context of this invention, therefore, before experimentalanimals are immunised, the target substance acting as a hapten is linkedto a high molecular weight carrier that is suitable for imparting to thetarget substance acting as a hapten the activity of a complete antigen.

By suitable carrier molecules there are to be understood within thecontext of this invention especially macromolecular compounds that havefreely accessible reactive groups for the linking reaction with thehapten and that are capable, by being linked to the hapten, of impartingto the latter an immunogenic potential or of reinforcing its existingimmunogenicity.

Special preference is given within the context of this invention tomacromolecular compounds containing freely accessible reactive aminogroups.

Very especially preferred for use according to the invention as carriermolecule are lysine-rich proteins having a molecular weight of from10,000 to 1,500,000, for example bovine serum albumin (BSA: MW 66,200),human serum albumin (HSA; MW 58,000) or keyhole limpet haemocyanin (KLH;MW>1,000,000), which are commercially available and are thus availablein any desired amount.

It is, of course, possible within the context of the present inventionalso to use other macromolecular compounds as carrier molecules providedthat they fulfil the above-mentioned requirements, such as porcinethyroglobulin, B2 microglobulin, haemocyanin, immunoglobulins, toxins(cholera, tetanus, diphtheria toxin, etc.), polysaccharides,lipopolysaccharides, natural or synthetic polyadenyl and polyuridylacids, polyalanine and polylysine polypeptides or cell-membranecomponents, for example formalin- or glutaraldehyde-treated erythrocytecell membranes.

Also suitable for use as carrier molecule in the method according to theinvention is, for example, the purified rabbit IgG fraction againstmouse IgG (H+L) according to the method described by H Kawamura and JABerzofsky (1986).

The conjugation of the hapten to the carrier molecule can be effectedeither directly or, preferably, by way of a spacer fragment which isoptionally first added onto the hapten molecule.

The linking of the substance to be analysed to the carrier molecule mustbe effected in such a manner that the relevant structural elements ofthe target substances remain freely accessible and are thus capable oftriggering a specific immune response, that is to say of inducing theformation of specific antibodies. In the preparation of the sulfonylureaderivatives, especially of the triasulfurone derivatives, care musttherefore be taken to ensure that those structural elements areretained.

Within the context of the present invention it has been found,surprisingly, that for a specific immune response to be triggered it isnot essential for the whole intact sulfonylurea molecule to be present,but that, on the contrary, selected fragments of the molecule also leadto excellent results and, with regard to the affinity of the resultingantibody, ultimately to even better results. For example, monoclonalantibodies having a high degree of affinity and selectivity towardsherbicides of the sulfonylurea class can be prepared using exclusivelythe sulfonamide moiety as linking component, instead of the complexwhole molecule consisting of sulfonamide moiety, urea bridge andheterocycle moiety, and linking that sulfonamide moiety, as describedhereinbefore, to a suitable carrier molecule. That is of great advantagefrom a technical point of view, since fragments of the molecule areeasier to handle than is the very much more complex whole molecule.

As shown within the context of the present invention, the immunisationof donor animals with the conjugate described hereinbefore consisting ofone of the customarily used high molecular weight carriers and thesulfonamide moiety of a sulfonylurea herbicide results in the productionof highly specific antibodies, which in the end allows the production ofmonoclonal antibodies that have on average a very much higher degree ofaffinity towards the particular target molecule than is the case whenthe whole molecule is used as linking component.

An important aspect of the present invention is thus the use of thesulfonamide moiety of sulfonylurea herbicides for the production ofconjugates that can be used for the immunisation of donor animals andthat lead ultimately to the production of monoclonal antibodies having ahigh degree of affinity towards said herbicide molecules. The saidsulfonamide moiety is preferably substituted arylsulfonyl, the arylradical normally being a phenyl, pyridyl, thienyl or pyrazolyl radical.Typical examples of sulfonylurea herbicides are described, for example,in EP 0 158 600 and EP 0 367 887 and in the literature referred totherein.

In a preferred embodiment of the present invention, therefore, it is notthe whole triasulfurone molecule that is linked to the carrier molecule,but rather only a selected moiety thereof that comprises the particulardesired determinant group in a form that triggers a very specific immuneresponse. Especially preferred within the context of this invention is atriasulfurone fragment that remains restricted to the sulfonamideportion of the triasulfurone.

The said sulfonamide portion may be obtained, for example, by cleavingthe corresponding sulfonylurea herbicide hydrolytically at thesulfonamide function using methods known per se. The sulfonamide linkingfragment obtainable in that manner thus has a terminal --SO₂ --NH₂ groupto which it is then very easily possible to link a suitable spacerfragment which can be used for the subsequent conjugation of the haptenmolecule. The linking of the spacer fragment can be achieved, forexample, by reacting the previously mentioned --SO₂ --NH₂ group with areactive carboxylic acid derivative, such as a succinic acid derivative,or preferably with the corresponding anhydride. When a succinic acidderivative is used the group --SO₂ --NH--C(O)--CH₂ CH₂ COOH is formed inthe process. The reaction is preferably carried out in a basic medium,especially in the presence of a suitable catalyst, such as the1,8-diazabicyclo[5.4.0]undec-7-ene used in Example 1.2.

The terminal carboxy grouping can also be convened using known methodsthat are frequently described in the literature into an amino or an SHgroup that likewise has a reactivity sufficient for linking the haptenmolecule.

Suitable spacer fragments for the conjugation of the hapten to thecarrier molecule are therefore especially compounds that contain atleast one or more reactive groups that are capable of interacting withthe freely accessible reactive groups of the carrier molecule.

Special preference is given within the context of this invention to theuse of spacer fragments comprising from 2 to 10 bridge carbon atoms andhaving as reactive group(s) one or more reactive groups, such as amino,carboxy or SH group(s). Those reactive groups may be reacted usingprocesses known per se with the reactive groups of the hapten moleculeand the carrier molecule to form a hapten-carrier conjugate.

It is possible, for example, to bind a spacer fragment by way of areactive amino group, using dialdehydes (for example glutaraldehyde), toone of the free amino groups of the carrier molecule. If the spacerfragment has a reactive SH group, the conjugation of the hapten to thecarrier molecule can be carried out by means of oxidation involving freeSH groups of the carrier.

Special preference is given within the context of this invention to theuse of spacer fragments having a carboxy group that can be linked withthe aid of water-binding agents, such as a carbodiimide, preferablyN,N'-dicyclohexylcarbodiimide, to a free amino group of the carriermolecule.

In order to link the antigen to the carrier protein, it is thereforeadvantageous first to produce a derivative capable of effecting thatlinking.

In a specific embodiment of the present invention the whole intactsulfonylurea molecule is used for the preparation of conjugates thatlead after immunisation of a donor animal to a specific immune responseand ultimately to the production of monoclonal antibodies tosulfonylurea herbicides of formula (A).

Triasulfurone derivatives that can be used as linking components withinthe context of the present invention are therefore especially those offormula (A), ##STR1## that have in the 4-position of the triazine ringan R--(CH₂)_(n) --O-- grouping, in which

R is COOH when n is an integer from 1 to 10, preferably from 1 to 6, and

R is COOH, NH₂ or SH, especially NH₂, when n is an integer from 2 to 10,preferably from 2 to 6.

It has, however, been found, surprisingly, that instead of the complexmolecules of formula (A) the considerably simpler fragments of formula(B) can be used as linking components.

In a preferred embodiment of the present invention, therefore, for thepreparation of conjugates that after immunisation of a donor animal leadto a specific immune response and ultimately to the production ofmonoclonal antibodies having a high degree of affinity towardssulfonylurea herbicides of formula (A) there is used the sulfonamidemoiety of said sulfonylurea herbicide. By convening that sulfonamidemoiety into the more stable fragment (B) there is obtained a virtuallyideal linking component that is outstandingly suitable for theproduction of monoclonal antibodies having a high degree of affinitytowards sulfonylurea herbicides of formula (A).

Especially preferred are triasulfurone fragments of formula (B),##STR2## which can be thought of as being formed by replacement of thetriazinamine grouping by an R'--(CH₂)_(n) -- group, wherein

R' is COOH, NH₂ or SH, especially COOH, and n is an integer from 1 to10, preferably from 1 to 6, and which are therefore restricted to thesulfonamide portion.

Preference is given within the context of the present invention to thetriasulfurone derivative of formula (IV) which, as described in detailbelow, can be prepared in a 4-step process. The starting compounds andreactants that can be used in that process are known or can be preparedby analogy with known, structurally similar compounds.

Especially preferred within the context of this invention is thetriasulfurone derivative of formula (V), which can be thought of asbeing formed by replacement of the triazinamine grouping by a--(CH₂)(CH₂)COOH grouping.

The resulting triasulfurone derivatives that are capable of linking arenovel and, by virtue of their specific linking ability, are a valuablestarting material in the production of monoclonal antibodies havingtriasulfurone specificity. They therefore constitute an important partof the present invention.

According to the triasulfurone derivative used, the actual linkingreaction is carded out using preferably the active ester method or thediazonium method [Kelly et al (1985)]. When the active ester method isused, the triasulfurone derivative is first solubilised in a suitablesolvent. Suitable solvents are especially aprotic solvents that have alow evaporation rate, such as N,N-dimethylformamide (DMF) or dimethylsulfoxide (DMSO).

The carboxy groups are then derivatised to form an active ester byreacting the previously solubilised triasulfurone derivative with, forexample, N-hydroxysuccinimide, N-hydroxysulfosuccinimide,N,N'-dicyclohexylcarbodiimide or N,N'-carbonyldiimidazole or withderivatives of those compounds.

The active ester is then separated off from the reaction mixture andadded to BSA or KLH. After an incubation period of from 0.1 to 12 hours,preferably from 4 to 5 hours, the precipitate is removed. Thesupernatant can then be used for the actual immunisation reaction, ifnecessary after further purification steps.

In addition to the active ester method preferred within the context ofthis invention, it is also possible to use alternative methods forlinking the hapten to the carrier molecule, for example the mixedanhydride method. In that method the carboxy group of the spacerfragment is linked to the carrier molecule using acetic anhydride or thecarbodiimide derivative 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide.

If the linking of the derivative takes place by way of a reactive NH₂group, it is preferable to use the diazonium method described by Kellyet al (1985).

In that method, sodium nitrite is added to the corresponding reactiveNH₂ group, preferably at reduced temperature, especially at atemperature of approximately 0° C., in strongly acidic solution. Theresulting diazonium salt is then added slowly to a buffered basicsolution of the carrier molecule. After further purification steps, thatsolution can then be used for the actual immunisation.

The immunisation of the donor animals is effected by means of one ormore administrations of the hapten linked to a high molecular weightcarrier molecule. Special preference is given to 2 or 3 administrations,at intervals of from 7 to 30 days, especially from 12 to 16 days.

The form of administration preferred within the context of the presentinvention is injection, which may be effected intravenously,intraperitoneally or subcutaneously. A combination of subcutaneous andintraperitoneal injection is preferred.

The antigen (triasulfurone conjugate) is present in that case in asuitable buffer, for example a PBS buffer, that contains one of theadjuvants customarily used. Within the context of this invention specialpreference is given to the use of Freund's adjuvant.

After a rest period of from 0.5 to 4 months a further singleadministration of the hapten conjugate in a dose of from 10 μg to 1000μg, especially from 50 μg to 500 μg, is given.

Within a period of from 1 to 6 days after the final dose, the donoranimals are sacrificed and a spleen cell suspension is prepared.

The isolated spleen cells are suspended in a suitable buffer (forexample a BSS buffer) and stored in the form of a cell suspension untilthey are fused with suitable myeloma cells.

Initially, those fusions were complicated by the fact that the myelomacell lines also synthesised monoclonal antibodies, with the result thatthe hydrid produced two types of monoclonal antibodies, one thatoriginated from the myeloma cell and a second that was determined by thegenetic information of the immunocompetent cell.

Within the context of the present invention, therefore, preference isgiven to the use of tumour cells that are not capable themselves ofproducing monoclonal antibodies, such as SP2/0-Ag14 (Shulman et al,1978), X63-Ag8.653 or PAI [Stocker et al (1982)], which very muchsimplifies the analysis of the resulting fusion products. For successfulfusion, it is advantageous for the spleen cells to be present in a 2 to20 fold excess in relation to the myeloma cells.

The fusion of spleen and myeloma cells is carried out in a specialfusion medium comprising a composition that provides optimum conditionsfor the intended cell fusion.

The said fusion medium is preferably a buffer solution that contains oneof the fusion promotors customarily used for the fusion of cells, forexample Sendai viruses or other paramyxoviruses, optionally inUV-inactivated form, calcium ions, surface-active lipids, for examplelysolecithin or polyethylene glycol. Especially preferred within thecontext of this invention is the use of polyethylene glycol (PEG),especially polyethylene glycol (PEG) having an average molecular weightof from 600 to 6000, in a concentration of from 30% to 60%. Especiallypreferred is PEG 4000 at a concentration of approximately 50%. Theoptimum fusion temperature is from 18° C. to 39° C. Especially preferredis a temperature of 37° C.

Once the fusion of the immunocompetent spleen cells with the myelomacells has taken place, the fused antibody-producing hybrid cells areselected using methods known per se.

Various possible methods exist for selecting successful fusion events(hybrid cells) from the two types of parent cells. Routinely, a millionor more cells of each parent type are used in the fusion protocol. Sincethe fusion does not take place with 100% frequency, it may become adifficult undertaking to separate the fusion products from the enormousnumber of non-fused parent cells and parent cells that have fused withthemselves.

As already mentioned hereinbefore, the hybridoma cells are formed byfusion of short-lived antibody-producing (spleen) B cells withlong-lived myeloma cells.

The desired result is a long-lived cell line that produces antibodies.Since the spleen cells have only a limited lifespan in culture, it istherefore possible in principle simply to wait until all the non-fusedspleen cells and all the spleen cells that have fused with themselveshave died. However, there then still remains the task of separating thelong-lived antibody-producing cells from the long-lived cells that donot produce antibodies.

A viable selection system is based on the availability ornon-availability of the enzyme hypoxanthine-guaninephosphoribosyltransferase (HGPRT). That enzyme is a constituent of thepurine salvage pathway in mammalian cells. Those cells are, moreover,also capable of synthesising purines de novo.

Under normal conditions the two methods of synthesis probably runparallel with one another to a certain extent.

If, however, a cell has no HGPRT, the salvage pathway is blocked and thepurines have to be prepared from non-purine material.

For the selection of HGPRT-negative myeloma cells, there are generallyused so-called purine antimetabolites, for example 8-azaguanine, whichis structurally very similar to the purine guanine and can thereforereplace the latter in some of its normal reactions.

Azaguanine is incorporated into DNA, which leads to impairment of normalgrowth behaviour and finally to the death of the cell. Since azaguaninehas to be replaced by way of the salvage pathway, all the cells thathave no HGPRT activity are unable to utilise azaguanine and thereforegrow in its presence.

A selective system that operates with the same enzyme but in reverse, inthat in this case HGPRT-positive cells are selected, is described in J.W. Littlefield (1964).

That selection system is based on the use of the so-called HAT mediumwhich comprises, inter alia, hypoxanthine, aminopterin and thymidine(HAT medium) as constituents. Aminopterin is an antimetabolite thatinhibits de novo purine synthesis and the methylation of deoxyuridylateto form thymidylate.

Hypoxanthine is able to act as an auxiliary purine in a case where theaminopterin blocks the de novo purine synthesis, while thymidine renderssuperfluous the necessity for the methylation of deoxyuridylate.

Thus in the presence of aminopterin all HGPRT-positive cellsproliferate, while the HGPRT-negative cells die.

In the hybrid system that is used for selection within the context ofthis invention, the myeloma cells are preferably resistant towardsazaguanine and sensitive towards aminopterin, that is to say they areHGPRT-negative. The antibody-producing cells, on the other hand, areHGPRT-positive.

By means of fusion of the cells and culturing in a HAT medium, the cellsthat have successfully fused with one another can be selected, since themyeloma cells that are responsible for the proliferation are able togrow only in the presence of an HGPRT activity, and that activity has tobe provided by the HGPRT-positive cell line.

Although the HGPRT-positive antibody-producing cell lines are not killedin that medium, they survive for only a certain amount of time and areunable to proliferate.

Thus as a result of the fusion of the cells in a HAT medium a system iscreated in which the myeloma cells and the antibody-producing cells areable to grow for a period of time sufficient for the production ofhybrid cells but in which only the hybrid cells are able to survive andproliferate.

In a special embodiment of the present invention the fused hybrid cellsare cultured in the presence of macrophages, so-called feeder cells,previously isolated from the peritoneum of untreated, non-immunisedexperimental animals. For the culturing and selection of the fusedhybrid cells, the cell suspension is divided into several aliquots andthe individual aliquots are examined continuously for the development ofhybrid cell cultures and for the formation of antibodies.

Especially preferred within the context of this invention is theculturing of the fused hybrid cells on microtitre plates.

The cell suspension obtained after fusion is divided amongst theindividual wells in a microtitre plate and cultured for a period of from7 to 30 days under conditions suitable for promoting the growth of thefused hybrid cells (for example HAT/HT media).

The supernatants of grown hybrid cultures are examined continuously forthe formation of antibodies.

Positive hybrid cell cultures are then separated by means of knownmethods, preferably using the limiting dilution method, and then clonedin suitable culture media.

The supernatants of the grown cell clones are likewise examined for theformation of antibodies.

The hybridoma cell clones according to the invention that have beenproduced in accordance with the foregoing description are screened forthe formation of suitable monoclonal antibodies, preferably using one ofthe immunoassays customarily used for that purpose, for example anenzyme-linked immunoassay or a radioimmunoassay.

In the enzyme-linked immunoassay the hapten conjugates characterised indetail hereinbefore are first adsorbed onto a solid carrier. Theremaining free binding sites are then saturated, and thus blocked, bythe addition of carrier molecules.

For the detection of monoclonal antibodies, aliquots of the supernatantsof said hybridoma cell clones are incubated with the carrier-boundhapten conjugates.

The present invention relates also to the production of monoclonalantibodies using methods that are known per se, wherein the hybridomacell lines according to the invention, characterised in detailhereinbefore, that synthesise the antibodies according to the inventionand preferably secrete them into the surrounding medium, or clones orsubclones thereof, are cultured in vitro or in vivo using known methods.

The in-vitro culturing of the hybridoma cells according to the inventionis effected in suitable culture media, especially in the standardculture media customarily used, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 Medium, which may optionally be supplementedby the addition of mammalian sera, for example foetal calf serum, or bygrowth-promoting additives and trace elements.

The isolation of the monoclonal antibodies preferably begins withprecipitation of the immunoglobulin fraction from the individualsupernatants of the hybridoma cultures, for example using ammoniumsulfate. Them follow further working-up and purifying steps that areknown to a person skilled in this field and that include, for example,the use of chromatographic methods, for example gel filtration,ion-exchange chromatography, DEAE-cellulose chromatography, protein A orimmunoaffinity chromatography.

Large amounts of the monoclonal antibodies according to the inventionmay, however, also be obtained using in vivo methods.

For example, it is possible to inject into suitable mammalsantibody-producing hybridoma cell clones that induce the development ofantibody-producing tumours in the treated animals. After a period offrom 1 to 3 weeks, the antibodies can be isolated from the body fluidsof the animals so treated.

In a special embodiment of the present invention, female Balb/c micethat have optionally been pretreated with a hydrocarbon, such aspristane, are injected intraperitoneally with a hybridoma cell cloneaccording to the invention.

From one to three weeks after the injection of the hybridoma cell clone,the ascites fluid is collected and stored until it is worked up further.

The isolation of the monoclonal antibodies is effected in a manner thatis precisely analogous to the isolation described hereinbefore from thesupernatants of hybridomas cultured in vitro.

The procedures described hereinbefore for the triasulfurone derivativescan of course be applied analogously also to other sulfonylureaderivatives.

The present invention relates also to the use of the antibodiesaccording to the invention in one of the customary immunoassays for thedetection of sulfonylurea herbicides, especially for the detection oftriasulfurone, and for the differentiation of sulfonylurea herbicides,especially triasulfurone, from structurally related compounds,especially from triasulfurone analogs selected from the group consistingof primisulfurone, sulfometurone-methyl, tribenurone-methyl,thiameturone-methyl, chlorosulfurone, bensulfurone-methyl,metsulfurone-methyl, nicosulfurone-methyl and DPX 9636, in soil, air andwater samples, and optionally in extracts from plants or otherbiological material.

The monoclonal antibodies according to the invention can thus be used inall known immunoassays that are based on the specific binding betweenantigen and the corresponding monoclonal antibody, for example in aradioimmunoassay (RIA), an enzyme-linked immunoassay (ELISA), animmunofluorescence test, etc.

In the RIA test, the monoclonal antibody according to the invention canbe used as such or in the form of a radioactively labelled derivative.All the modifications of the RIA test known hitherto can be used for thedetection of the target substances relevant within the context of thisinvention, for example an RIA test in homogeneous or solid phase, aheterogeneous RIA and a simple (sandwich) RIA test with direct orindirect (competitive) detection of the antigen. The same applies alsoto the use of an enzyme-linked immunoassay.

Preference is given within the context of this invention to the use of amonoclonal antibody according to the invention in a competitiveimmunoassay for the detection of triasulfurone.

The principle of the competitive immunoassay is based on a competitionbetween a labelled antigen or an antigen bound to a solid carrier and afree antigen for the relevant binding sites on the antibody molecule.

There are, in principle, two possible methods of carrying out thatcompetitive immunoassay.

a) The first method is based on the competition between the antigenbound to a solid carrier and free antigen for the free binding sites onthe antibody, which is provided with a marker. The binding of theantigen to a solid carrier can be effected either directly or by way ofa carrier molecule.

The concentration of free antigen is determined in this case by means ofthe decrease in the labelled antibody bound to the carrier-fixedantigen. The decrease is proportional to the amount of free antigencontained in the sample.

b) An alternative method is based on the fact that free antigen andlabelled antigen compete with one another for the relevant binding siteson the antibody, which in this case is bound to a solid carrier.

The concentration of free antigen is determined by means of the decreasein labelled antigen, which varies as a function of the concentration offree antigen.

Examples of solid carrier material that is suitable for binding theantigen or the antibody are the plastics surface of a microtitre plateor a test tube, the surface of balls of polystyrene, polypropylene,polyvinyl chloride, glass or plastics, or the surface of strips offilter paper, dextran cellulose or nitrocellulose, or similar materials.Those materials are coated with one of the monoclonal antibodiesaccording to the invention or an antigen, it being possible for thebinding to the carrier material to be effected by simple adsorption oroptionally after prior activation of the carrier material with, forexample, glutaraldehyde or cyanogen bromide.

Special preference is given within the context of this invention to theuse of the monoclonal antibody according to the invention in anenzyme-linked immunoassay [ELISA (enzyme linked immunosorbent assay)].In that immunoassay the monoclonal antibody according to the inventioncan be used as such or in the form of an enzyme-linked derivative.

The ELISA assay is based either on the use of an enzyme-linkedderivative of the antibody according to the invention or ofenzyme-linked antibodies known per se that recognise an epitope of anantibody according to the invention and bind thereto.

Special preference is given within the context of this invention to theuse of an ELISA assay in which one of the carrier materials describedhereinbefore is first coated with an antigen. The carrier-bound antigenis then incubated with a test solution that contains the antigen to bedetected and one of the antibodies according to the invention. Theantigen to be detected may be present either in free form or as aconstituent of a water or soil sample.

After an incubation period of from 10 minutes to 2 hours, the wholebatch is incubated with an enzyme-labelled antibody that recognises themonoclonal antibody according to the invention and binds to the latter.An example of such an enzyme-labelled antibody is a phosphatase-labelledgoat anti-sheep immunoglobulin, or a corresponding goat anti-mouseantibody, which are commercially available. The amount of bound antibodyprotein can be determined with the aid of an enzyme-substrate reaction,for example by means of spectroscopic methods.

Also preferred within the context of this invention is an ELISA testthat is based on the competition between labelled and free antigen forthe antibody bound to one of the carrier materials mentionedhereinbefore.

The amount of free antigen present in a specific sample is determined inthis case by means of the decrease in labelled antigen, which is themore precise the more free antigen the sample contains.

The present invention relates further to means for the qualitative andquantitative determination of sulfonylurea herbicides, especially oftriasulfurone, in the form of a test kit that may comprise, in additionto the monoclonal antibodies according to the invention and/or theirderivatives, optionally also other monoclonal or polyclonal antibodies,especially labelled monoclonal or polyclonal antibodies, and furtheradditives.

Special preference is given within the context of this invention to testkits based on one of the immunoassays customarily used, selected fromthe group consisting of radioimmunoassay, enzyme-linked immunoassay andchemiluminescence assay. Especially preferred are test kits in which thedetection of the target substance is based on a competitive immunoassay,especially on an enzyme-linked immunoassay (ELISA).

Test kits for the radioimmunological detection of triasulfurone that arepreferred within the context of this invention may comprise, forexample, the following components:

(a) a suitable carrier material that may be uncoated or coated with oneof the antibodies according to the invention or with an antigenconjugate;

(b) optionally freeze-dried or concentrated solutions of one of theantibodies according to the invention and/or of a radioactively labelledderivative thereof, or radioactively labelled antigen or standardsolutions of the antigen;

(c) buffer solutions and

(d) optionally polypeptides, detergents and other additives that, forexample, prevent non-specific adsorption and aggregate formation and

(e) pipettes, reaction vessels, calibration curves, packaging labelsetc.

Test kits for the immunological detection of triasulfurone that arebased on an enzyme-linked immunoassay (ELISA) may comprise, for example,the following components:

(a) a suitable carrier material that may be uncoated or coated with oneof the antibodies according to the invention or with an antigenconjugate;

(b) optionally freeze-dried or concentrated solutions of one of theantibodies according to the invention and/or of a second enzyme-labelledmonoclonal or polyclonal antibody that is directed against the antigento be determined or against an antibody that recognises the antigen;

(c) enzyme substrates in solid or dissolved form;

(e) the antigen or standard solutions of the antigen;

(f) buffer solutions;

(g) optionally polypeptides, detergents and further additives that, forexample, prevent non-specific adsorption and aggregate formation and

(h) pipettes, reaction vessels, calibration curves, colour charts,packaging labels, etc.

A test kit for the detection of triasulfurone that is based on achemiluminescence test may comprise, for example, the followingcomponents:

(a) a suitable carrier material that may be uncoated or coated with oneof the antibodies according to the invention or with an antigenconjugate;

(b) optionally freeze-dried or concentrated solutions of one of theantibodies according to the invention and of a second polyclonalantibody that is capable of recognising the first antibody according tothe invention and is linked to a chemiluminescent marker,

(c) solutions comprising a component that triggers the emission oflight, for example H₂ O₂ and NaOH;

(d) buffer solutions;

(e) optionally polypeptides, detergents and other additives that preventnon-specific adsorption and aggregate formation and

(f) pipettes, reaction vessels, packaging labels, etc.

Carrier materials that may be used within the context of the presentinvention comprise especially insoluble, polymeric materials, selectedfrom the group consisting of polystyrene, polyethylene, polypropylene,polyester, polyacrylonitrile, polyvinyl chloride, polyacrylamide,nitrocellulose, cross-linked dextran, fluorinated resins, agarose,cross-linked agarose, polysaccharides, etc. Other materials are alsoconceivable, however, for example glass, metal, nylon-based netting,etc.

The carrier materials mentioned individually hereinbefore may be in verydifferent forms and, depending on the specific intended use, may be in awide variety of shapes. Those include, for example, dishes, balls,plates, small rods, cells, small bottles, small tubes, fibres, nets,etc.

Frequently used in the manufacture of test kits are, for example,microtitre plates made of transparent plastics materials, for examplepolyvinyl chloride or polystyrene, that may be uncoated or coated withone of the antibodies according to the invention, free antigen or anantigen conjugate. Also used are small balls, tubes or rods made ofpolystyrene and polystyrene latex, it being possible to separate thesurrounding latex material from the polystyrene particles by means ofcentrifugation.

A further component of the test kit according to the invention aremarkers or indicators by means of which it is possible to detect thepresence of a complex-forming reaction, especially an immune reaction,that preferably results in an antigen/antibody complex or in aligand/receptor complex, it being possible optionally to makequantitative as well as qualitative statements regarding the antigen tobe detected. Suitable markers or indicators are both individual atomsand molecules that may participate either directly or indirectly in thegeneration of a detectable signal. Those markers or indicators may belinked directly to the antigen to be detected or to one of themonoclonal antibodies according to the invention or may be incorporatedtherein. They may, however, alternatively be in the form of individualsubstances or in the form of a constituent of a separate compound thatis neither the antigen to be detected itself nor one of the monoclonalantibodies according to the invention, but that is capable for its partof reacting with the receptor molecule, for example in the form of acomplex formation.

Those separate compounds are preferably a second antibody molecule thatmay be of monoclonal or of polyclonal origin, a complement protein orfragments thereof, S.aureus protein A, etc. Those separate compoundsrecognise and bind specifically to a receptor molecule, for example theantigen to be detected or one of the monoclonal antibodies according tothe invention, but preferably to a receptor molecule that is present inthe form of a complex.

In many cases other, additional reagents are required that lead to adetectable signal only in cooperation with the marker. That is the caseespecially when enzymes are involved.

Markers or indicators that may be used within the context of the presentinvention are very well known to a person skilled in the art ofimmunology and immunochemistry. They include, for example, radioactivelylabelled elements or substances, enzymes or chemiluminescent substances.The following fist of possible markers or indicators is intended merelyto illustrate by way of example the great variety of the substances andreagents that may be used, without thereby limiting the subject of theinvention in any way.

Suitable markers or indicators are to be found, for example, amongst thegroup of the radioactive elements. Preference is given especially toelements that either emit γ rays themselves, for example ¹²⁴ I, ¹²⁵ I,¹²⁸ I, ¹³² I, ⁵¹ Cr, or that induce the emission of those rays, forexample ¹¹ C, ¹⁸ F, ¹³ N. Also suitable are so-called β-emitters such as¹¹¹ In, ¹⁴ C and ³ H.

Further suitable markers include chemiluminescent substances, especiallyfluorescent substances, which can be very easily bound chemically to theantigen or an antibody without denaturing the latter. The resultingfluorochrome can be detected very easily by means of fluorometricmethods. There may be mentioned individually here fluorochromes selectedfrom the group consisting of fluorescein isocyanate, fluoresceinisothiocyanate, 5-dimethylamino-1-naphthalenesulfonyl chloride,tetramethylrhodamine isothiocyanate, lissamine, rhodamine 8200 sulfonylchloride, etc.

Other fluorescent agents and a description of analytical techniques areto be found in DeLuca, "Immunofluorescence Analysis", in: Antibody As aTool, Marchalonis et at, John Wiley & Sons, Ltd., pp 189-231 (1982).

Special preference is given within the context of this invention to theuse of enzymes as marker or indicator substances, for examplehorseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucoseoxidase, glucoamylase, carboxylic acid anhydrase, acetylcholineesterase, lysozyme, malate dehydrogenase, glucose 6-phosphatedehydrogenase, etc. When using enzymes as marker substances it isnecessary to add additional reagents that allow the formation of animmune complex to be followed by way of the enzyme activity andoptionally a stop reagent that can be used to terminate the enzymereaction.

Special preference is given to reagents that lead to a colour reaction.In the case of horseradish peroxidase there may be mentioned by way ofexample hydrogen peroxide which, in combination with an additional,oxidised dyestuff precursor such as diaminobenzidine oro-phenylenediamine, leads to a brown or yellow colouring. When usingglucose oxidase as marker substance it is possible, for example, to use2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonic acid) [ABTS] assubstrate.

The present invention therefore relates further to the use of test kitsthat comprise as reagent at least one of the monoclonal antibodiesaccording to the invention for the rapid and effective, qualitativeand/or quantitative detection of sulfonylurea herbicides, especially oftriasulfurone, and for the differentiation of those compounds fromstructurally related compounds, especially from triasulfurone analogs,selected from the group consisting of metsulfurone-methyl,bensulfurone-methyl, thiameturone-methyl, sulfomethurone-methyl,tribenurone-methyl, chlorosulfurone and primisulfurone.

I. NON-LIMITING EXAMPLES EXAMPLE 1 Synthesis of Triasulfurone LinkingComponents

1.1: Preparation of a triasulfurone derivative capable of linking to acarrier protein

The preparation of the triasulfurone derivatives capable of linking to acarrier protein can be carried out in accordance with the followingreaction scheme: ##STR3##

In detail, the four-step process shown above is carried out as follows:

Step 1: Preparation of 2-hydroxypropylcarbamoyloxymethyl benzene

22.5 g of 3-amino-1-propanol (Aldrich) and 30.3 g of triethylamine areintroduced into 400 ml of methylene chloride and then within a period of30 minutes at from 15° C. to 20° C. with slight cooling 51.2 g of1-chloroformic acid benzyl ester are added. The solution so obtained isthen stirred for a further 2 hours at room temperature, washed 3 timeswith deionised water, dried with Na₂ SO₄ and totally concentrated.

The crude product is then purified by chromatography over a silica gelcolumn (300 g). The eluant used is a 1:1 (v/v) mixture of toluene andethyl acetate. The eluate is crystallised from petroleum ether (fraction30° C. to 70° C.). After filtering and drying, the compound of formula(I) is obtained in a yield of 13 g (20.7% of the theoretical yield);melting point 52° C.-53° C.

Step 2:

1.85 g of trimethylamine, dissolved in 20 ml of acetone, are addedwithin the course of one minute to a suspension consisting of 4.12 g of2-amino-4-chloro-6-methyltriazine, 3.9 g of potassium carbonate and 6.3g of a compound of formula (I)[2-hydroxypropylcarbamoyloxymethylbenzene] in 80 ml of acetone. Themixture is then stirred for a further 15 hours at a temperature of from40° C. to 45° C. before being concentrated to dryness by evaporation andtaken up in ethyl acetate.

After washing twice with deionised water, the ethyl acetate phase isdried over Na₂ SO₄ and concentrated to a residual volume of a few ml.The resulting crystals are filtered off, washed and dried, affording thecompound of formula (II) (white crystals) in a yield of 3.1 g (34.2% ofthe theoretical yield); melting point 126° C.-128° C.

Steps 3 and 4: Preparation of3-(6-(3-amino-n-propoxy)-4-methyl-1,3,5-triazin-2-yl)-1-[2-(2-chloroethoxy)-phenylsulfonyl]-urea

2.75 g of 2-(2-chloroethoxy)-phenylsulfonyl isocyanate and 3.35 g ofaminotriazine of formula (II) are stirred in 20 ml of dioxane (abs.) for5 hours at a temperature of from 80° C. to 85° C. The clear solution isconcentrated to dryness by evaporation and the crude product ischromatographed over silica gel using toluene:ethyl acetate=1:2 (v/v).

The fractions containing the sulfonylurea of formula (III) are combinedand hydrogenated with hydrogen directly in 200 ml of deionised water and1.83 g of Na₂ CO₃, in the presence of 0.5 g of Pd/C 5%. After filteringoff the catalyst, the colourless solution is acidified to a pH of from5.5 to 6.0 by the addition of 2N HCl. The precipitate thus formed isfiltered off and dried. The end product of formula (IV) is obtained in ayield of 2.6 g; melting point 146° C.-147° C.

The starting compounds and reactants used in the four-step processdescribed above are known or can be prepared by analogy with knownprocesses.

EXAMPLE 1.2 Preparation of a Triasulfurone Fragment Capable of Linkingto a Carrier Protein

The preparation of a second triasulfurone fragment capable of linking toa carrier protein can be carried out in accordance with the followingreaction scheme: ##STR4##

In detail, the following process steps are carried out.

Over a period of half an hour, 18.5 g of1,8-diazabicyclo[5.4.0]undec-7-ene, dissolved in dioxane, are addeddropwise at room temperature, with slight cooling, to a solution of 14.1g of 2-(2-chloroethoxy)phenylsulfonamide and 6.0 g of succinic anhydridein 150 ml of dioxane.

The resulting white suspension is stirred at room temperature for afurther 2 hours, acidified with 65 ml of 2N HCl and concentrated todryness by evaporation. The oily residue is taken up in ethyl acetateand washed twice with deionised water. After drying over Na₂ SO₄ andconcentrating again by evaporation, recrystallisation from 40 ml ofethanol yields 7.1 g (35.3% of the theoretical yield) of white crystalsof the compound of formula (V) having a melting point of 166° C.-168° C.

EXAMPLE 1.3 Triasulfurone-protein Conjugate

The triasulfurone derivative prepared in accordance with Example 1.3.1or the triasulfurone fragment prepared in accordance with Example 1.2 isconjugated either to bovine serum albumin (BSA; Fluka) or to keyholelimpet haemocyanin (KLH; Calbiochem) using the diazonium methoddescribed in Kelly et al (1985) or the activated ester method (Kulkarniet al., 1981).

1.3.1: Linking of the compounds of formula (IV) to BSH or KLH using thediazonium method

100 mg of the triasulfurone derivative of formula (IV) are suspended ina mixture of acetic acid (10 ml) and propionic acid (5 ml). Theresulting mixture is cooled to 0° C. and concentrated HCl (0.2 ml) andsodium nitrite (0.05 g) are added. The temperature of the reaction batchis kept constant at 0° C. for a period of 30 minutes. The resultingyellow solution is then divided into two 7.5 ml portions, each of whichis added slowly to 0.1 g of BSH or KLH in 20 ml of 0.05M borate buffer(pH 9.0). The pH of the solution is kept constant at 9.0 by the additionof 1M NaOH.

Before the end product of this reaction is used for immunisation, it isdialysed fully against a phosphate-buffered salt solution [(PBS) 0.01Msodium phosphate and 0.145M NaCl, pH 7.0].

1.3.2: Linking of the compound of formula (V) to BSH or KLH using theactivated ester method

In detail, the carboxy group of the triasulfurone fragment of formula(V) is solubilised at room temperature in N,N-dimethylformamide (DMF)(7.2 mg/200 μl) and then a 4 molar excess of α-hydroxysuccinimide (9.1mg/200 μl) and N,N'-dicyclohexylcarbodiimide (16 mg/200 μl) is added.The reaction mixture is stirred first for one hour at 22° C. and thenfor 18 hours at 4° C.

The precipitate formed during the reaction is removed by centrifugationfor three minutes at 12,000 g at room temperature and the activatedester is then added to BSA [12 mg] or KLH [12 mg] that has previouslybeen solubilised in 3.3 ml of a phosphate-buffered salt solution (PBSbuffer).

After incubation for 4 hours at a temperature of 4° C. the precipitatethat has formed is removed by centrifugation for 10 minutes at 2,000 gat 4° C. and the supernatant that remains is dialysed fully against PBSbefore being used for the immunisation experiments.

The extent of the linking reaction is determined by absorptionspectrophotometry. The molar ratio of triasulfurone fragment to BSA isapproximately 10:1, preferably 12:1.

EXAMPLE 2 Immunisation

2 groups of 5 four- to six-week-old female Balb/c mice (TierfarmSisseln, Switzerland) receive at intervals of 2 weeks a series of 3intraperitoneal and subcutaneous injections of KLH-conjugatedtriasulfurone. The dosage for the triasulfurone conjugate of formula(IV) prepared in Example 1.3.1 is 80 μg/injection, while thetriasulfurone conjugate of formula (V) prepared in Example 1.3.2 isadministered at a dosage of 60 μg/injection.

The first injection contains 0.1 ml of the conjugate in PBS, which hasbeen mixed in a ratio of 1:1 with 0.1 ml of complete Freund's adjuvant.

50 μl of that injection solution are injected intraperitoneally, and theremaining 150 μl subcutaneously.

In the second and third injections in the series, which take place 14and 30 days, respectively, after the first administration, the completeFreund's adjuvant is replaced with incomplete Freund's adjuvant.

One week after the final injection, blood serum is taken from theexperimental animals and the blood titre is determined by means of anELISA test, the microtitre plates having previously been coated withBSA-conjugated hapten (see Section 6).

After a rest period of 2 months, a further single intraperitonealinjection of the KLH conjugates is carried out at a dosage of 830 μg/200μl of PBS [triasulfurone conjugate of formula (IV)] or 540 μg/200 μl ofPBS [triasulfurone conjugate of formula (V)]. From three to four dayslater the mice are sacrificed and the spleen cells isolated from themare fused with the myeloma cell line PAI [Stocker et al (1982)] [seeExample 3.4].

EXAMPLE 3 Fusion Protocol

3.1. Obtaining feeder cells (peritoneal macrophages).

Untreated Balb/c mice approximately six to eight weeks old aresacrificed one day before the intended fusion and sterilised byimmersion in 70% alcohol.

The skin and the outer peritoneum are cut under sterile conditionswithout damaging the peritoneum. Using a sterile 5 ml plastics syringeand a sterile No. 18 injection needle, 4 ml of BSS (without Ca²⁺ andMg²⁺) and 1 ml of air are injected into the abdominal cavity.

After light massage of the abdomen (syringe and needle remaining in theabdominal cavity), the previously injected BSS buffer is drawn off fromthe peritoneum and introduced into a sterile Falcon tube. This procedureis repeated twice. The macrophages so obtained are cooled with ice andthen washed twice with 20 ml of BSS each time.

The macrophages are centrifuged for 10 minutes at 300 g and atemperature of 5° C. The pellet is then resuspended in 50 ml of HATmedium and the cell suspension is divided among 4 Costar plates having atotal of 24 wells (0.5 ml/well).

The macrophages thus prepared are then stored in an incubator at atemperature of 37° C. and a CO₂ concentration of 6%.

Approximately 4×10⁶ macrophages are required per fusion.

3.2. Culturing of the myeloma cell line PAI

The mentioned myeloma cell line PAI is a myeloma cell line that does notitself secrete any antibodies and that is described in Stocker et al.(1982).

50 ml of a well-grown culture comprising at least 10 million cells arerequired per fusion. The culturing of the myeloma cells is carded outpreferably in T 175 Falcon bottles (Messrs. Beckton & Dickenson).

One day before the fusion the culture medium (RPMI 1640) is replacedwith fresh RPMI 1640 medium. On the day of the fusion the PAI cells areharvested, introduced into a sterile 50 ml plastics tube and centrifugedfor 10 minutes at 300 g and at a temperature of 5° C. (MSE centrifuge,Chilspin model, UK). After the centrifugation the supernatant is drawnoff and discarded. The cells are washed twice with approximately 30 mlof BSS buffer (free of Ca²⁺ and Mg²⁺) each time (10 minutes at 300 g, 5°C.) and then resuspended in 5 ml of BSS.

An aliquot of the cell suspension is removed and stained withfluorescein diacetate (FDA) in order to determine the number of cells.The myeloma cells are stored on ice until they are used further.

3.3. Preparation of a spleen-cell suspension

The removal of the spleen from a Balb/c mouse previously immunised inaccordance with Example 2 is effected under sterile conditions and whilecooling with ice.

The previously immunised Balb/c mouse is sacrificed by breaking its neckand the spleen is removed under sterile conditions. For that purpose themouse is immersed briefly in 70% ethanol and dissected using sterileinstruments. The spleen is removed carefully and laid on a fine nylonnet, where it is finely chopped using scissors and then pressedcarefully through the net with the aid of a 5 ml syringe plunger withoutdestroying too many cells in the process. During the entire operationthe net is rinsed with BSS.

The cell suspension so obtained is introduced into 50 ml plastics tubesand centrifuged for 10 minutes at 300 g and at a temperature of 5° C.(MSE centrifuge, Chilspin model; UK). The cells are then washed twicewith 20 ml of BSS each time (10 minutes; 300 g; 5° C.; MSE Chilspin) andafter centrifugation the cell pellet is resuspended in 10 ml of BSS.

Until the fusion with PAI myeloma cells the spleen cells are kept onice.

3.4. Fusion: spleen cells and PAI myeloma cells

The ratio of myeloma cells to spleen cells for the fusion should be1:10.

Spleen cells (in BSS buffer) and PAI myeloma cells (in BSS buffer) areadded together in the given ratio and centrifuged for 10 minutes at 300g and at a temperature of 5° C. (MSE centrifuge, Chilspin model). Thepellet is resuspended in BSS buffer and the suspension is thencentrifuged again. The pellet is broken up by stirring carefully andplaced in a water bath at 37° C. 1 ml of preheated sterile PEG-4000(MERCK) is then added dropwise to the cells over a period of 60 seconds,the whole batch being agitated constantly. The cells are then shaken fora further 30 seconds before 5 ml of a previously heated BSS buffer(without Ca²⁺, Mg²⁺) are likewise added dropwise thereto over a periodof approximately 5 minutes with constant stirring.

The cells fused in the manner described are then centrifuged off (10minutes; 300 g; 20° C., MSE centrifuge, Chilspin model) and thesupernatant is drawn off and discarded. The cell pellet is resuspendedin 50 ml of HAT medium and the resulting cell suspension is dividedamong the 4 prepared Costar plates (microtitre plates having 24 wells,diameter per well 24 mm; total surface area for cell growth 2.0 cm²)(0.5 ml/well).

The Costar plates are incubated at a temperature of 37° C. and at a CO₂concentration of 6%.

EXAMPLE 4 Culturing the Hybrid Cells

On the first day after the cell fusion, 1 ml of HAT medium per well isadded to the culture plates. From 3 to 4 days after the cell fusion thefused cells are examined under a microscope. At the same time the spentmedium is removed by suction and replaced with 1 ml of fresh HAT medium.After a further 3 days (6-7 days after the cell fusion) the culturemedium is changed again. From the 7th to 10th day after the cell fusioneach well is examined for hybrids under a microscrope and the medium isreplaced from 2 to 3 times weekly.

As soon as hybrids have grown in a well, usually after from 2 to 4weeks, the HAT medium in that well can be replaced with HT medium. Thesupernatant of grown hybrid cultures (at least 10% of the well) isremoved using a sterile Pasteur pipette and tested for the presence ofantibodies.

As soon as a well is full of positive hybrid colonies, the latter can betransferred to new Costar plates in RPMI 1640 medium, the contents of afull well being divided among 2 or 3 new wells.

EXAMPLE 5 Cloning the Positive Hybrid Cells

The cells in a positive well are detached with the aid of a pipette andtransferred in 1 ml of medium into a tube. An aliquot is then removedand stained with FDA (dilution 1:2 with FDA: 50 μl cells+50 μl stain) inorder to determine the number of cells. The preferred number of cells isfrom 10⁵ to 10⁶ cells/ml. The hybrid cells are then diluted with HTmedium in a ratio of 1:100 (for example 100 μl of cells+9.9 ml of HTmedium).

25 ml of HT medium are introduced into each of two 50 ml Falcon tubesand made up to a total of 30 ml per tube with 5 ml of a macrophagesuspension. The macrophages have previously been isolated from a mouseand resuspended in 10 ml of HT medium (see Section 3.1).

In those Falcon tubes containing the macrophages the hybrid cells arediluted until a cell density of (i) 270 cells/30 ml and (ii) 90 cells/30ml, respectively, has been reached. These batches are then divided amongCostar plates (microtitre plates having 96 wells), 200 μl beingintroduced per well. This corresponds to a cell count of (i) 1.8cells/well and (ii) 0.6 cells/well, respectively. 1.5 microtitre platesare thus required per dilution.

After 7 days the individual wells are examined under a microscope andthe wells that contain cell clones are recorded. The dilution at whichapproximately 50% of the wells contain cell clones is used for the ELISAtest. This should generally be a dilution of 0.6 cells/well. Afterapproximately from 7 to 10 days the supernatants of the positive wells(with clones) are tested in an ELISA test for the presence of monoclonalantibodies and the positive clones are propagated on Costar plates(having 24 wells) in RPMI 1640 medium. Aliquots of those positive clonesare stored in liquid nitrogen.

EXAMPLE 6 Hybridoma Screening (ELISA Test)

First of all 100 μl of a solution of BSA-conjugated hapten [2 μg/ml] insodium carbonate buffer (50 mM, pH 9.6) are introduced into theindividual wells in a microtitre plate and the batch is incubatedovernight at 4° C. in a humidity chamber. Each of the wells is thenwashed five times with a 0.1% PBS Tween buffer. In order to block theunoccupied binding sites on the microtitre plate, 200 μl of a PBS-BSAsolution (1%) are introduced into each well. The batch is incubated for1-2 hours at room temperature and then washed with a 0.1% PBS Tweenbuffer.

200 μl of the hybridoma supernatant diluted in a ratio of 1:2 with PBSTween (0.1%) are then introduced into each well and the whole batch isincubated for 2 hours at room temperature. The wells are then washedagain five times with a 0.1% PBS Tween buffer.

There follows incubation with phosphatase-conjugated goat anti-mouseantibody (Kirkegaard & Perry Lab.). There are first added to each well100 μl of a goat antibody to mouse IgG, purified by affinitychromatography, that is present in a 1:1500 dilution in PBS Tween (0.1%)(Kirkegaard & Perry Laboratories) and is labelled with alkalinephosphatase.

The incubation period is 1.5 hours at room temperature. The individualwells are then washed again with PBS Tween (0.1%) (five times).

150 μl of a substrate-containing solution (1 mg/ml of p-nitrophenylphosphate) are then introduced into each well. After an incubationperiod of 2 hours in the dark, spectroscopic determination is carriedout at 405 nm. Positive hybridoma cells that secrete a specific antibodyemit a strong positive signal at the chosen wavelength. Those cells arethen cloned using the limiting dilution method [Godings (1980)]. Puremonoclonal antibodies can be obtained from the ascites fluid of suitablypretreated mice [Campbell AM, (1984)]

EXAMPLE 7 Expansion of Hybridoma Cells in the Mouse

In order to stimulate the production of ascites, female Balb/c mice(20-25 mg) (Tierfarm Sisseln, CH) are pretreated with 0.3 ml of pristaneoil (Aldrich Chemical) which is injected intraperitoneally. From 1 to 3weeks after the administration of pristane, the mice receive a secondinjection (0.2 ml of pristane oil, i.p.). At the same time as thissecond injection the animals receive 2×10⁶ hybridoma cells in 0.2 ml ofPBS.

The ascites fluid resulting from that treatment is collected,centrifuged at 800 g and storm at a temperature of -20° C. After thawingthe ascites fluid is centrifuged for 1 hour at 30,000 g. The topmostlayer, which comprises predominantly lipids, is removed. The proteinconcentration is then determined and adjusted to a value of 10 mg/ml bythe addition of PBS.

The immunoglobulin G fraction (IgG) is precipitated by the dropwiseaddition of 0.9 parts by volume of a saturated ammonium sulfate solutionat 0° C. After 1 hour the IgG fraction is pelleted by centrifugation forone hour at 22,000 g. The pellet is then dissolved in 20 mM tris-HClbuffer, pH 7.9, comprising 50 mM NaCl, and dialysed against the samebuffer overnight at 4° C. Further working up of the IgG fraction iscarried out by means of anion-exchange chromatography on a DE-52diethylaminoethylcellulose (Whatmann) column. The sample is diluted 1:2(v/v) with 20 mM tris-HCl, pH 7.9, until a final NaCl concentration of25 mM has been reached, and 10 mg of protein/ml of gel are applied tothe column. Elution is achieved by increasing the NaCl concentrationfrom 25 mM to 200 mM (linear gradient). In general, the elution ofmonoclonal antibodies takes place in the region of 80 mM NaCl.

The fractions are dialysed against PBS overnight at a temperature of 4°C. and stored at -70° C. The degree of purity is determined by means ofsodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), andby isoelectric focusing.

In the present case the degree of purity is >90%.

EXAMPLE 8 Triasulfurone Detection

The detection of triasulfurone is effected by means of a two-stepcompetitive ELISA test.

BSA-conjugated hapten, prepared in accordance with Example 1.3.1 or1.3.2, in a 50 mM sodium carbonate buffer (pH 9.6) (200 ng ofBSA-conjugated hapten/100 μl of sodium carbonate buffer) is first of alladsorbed on microtitre plates (for example Dynatech, type M 129A) andincubated overnight at a temperature of 4° C.

The plates are then washed five times with PBS buffer enriched with 0.1%(v/v) polysorbate 20 (Tween 20) (PBS Tween).

The remaining free binding sites of the solid carrier material are thenblocked by the addition of PBS-BSA in the form of a 1% (w/v) solution.After incubation for two hours at 22° C. the plates are washed againwith PBS Tween (0.1% ).

50 μl of the supernatant of the previously cloned hybridoma cells (in adilution of 1:2000 or 1:4000) or 50 μl of the previously purifiedmonoclonal antibodies (40-120 ng/ml) are incubated a) with 950 μl of astandard solution containing an increasing amount of triasulfurone or oftriasulfurone analogs, b) with triasulfurone-containing water samples orc) with triasulfurone-containing soil extracts. [All dilutions arecarried out in PBS Tween (0.1%)].

After an incubation period of 1 hour at room temperature (22° C.), 200μl of the antigen/antibody mixture are added to each well in themicrotitre plate and the whole batch is incubated for a further onehour. The wells are then washed five times with PBS Tween (0.1%) andcharged with 100 μl/well of goat anti-mouse IgG antibody that isconjugated to alkaline phosphatase (dilution 1:1500), and incubated fora period of 1.5 hours.

After washing again, 150 μl/well of the substrate p-nitrophenylphosphate dissolved in 1 mg/ml of diethanolamine buffer (1 mM, pH 9.8,enriched with 0.5 mM MgCl₂ ×6H₂ O) are added to the wells.

After an incubation period of 2 hours at a temperature of 22° C., acolour change is observed that is proportional to the amount of antibodythat has reacted with the antigen bound to the solid phase. Theintensity of the colour reaction that has occurred is determined at awavelength of 405 nm. The dilutions of the individual samples are sochosen that without the addition of an inhibitor (Bo) absorption valuesin a range of from 0.3 to 0.5 are obtained. For the controls (withoutantibody) values of A₄₀₅ ≦0.01 are obtained. All the samples aredetermined in triplicate.

In order to determine the amount of triasulfurone present in a sample,first of all a calibration curve is produced (FIG. 1 ), B/Bo×100 beingplotted against the concentration of inhibitor. (Bo represents theabsorption capacity measured without the addition of a triasulfuroneinhibitor to the antibody, and B is the absorption capacity whentriasulfurone inhibitors at various concentrations are added). The I₅₀value indicates the concentration of the antigen at which the binding ofantibody to the solid phase is inhibited by 50%. The I₅₀ value isdetermined using an ENZFITTER (Leatherbarrow, Elsevier-Biosoft)curve-calculation program based on a four-parameter logistical curve(Raab GM, 1983) and specially adapted to the prevailing conditions. Thequantitative determination of triasulfurone in soil or water sampleswithin the context of ELISA is also carried out using the ENZFITTERprogram, the adaptation of the curve being based on standards that runon any microtitre plate.

EXAMPLE 8.1 Analysis of Soil Samples

Aliquots (2 g) of standard soil samples of different origins areextracted in accordance with the methods described below:

Method (A) [according to Iwanzik and Egli (1989)]: A 100 g sample isextracted for 2 hours in an extractor by shaking with 300 ml of a 2:1mixture (v/v) of methanol and an aqueous phosphate buffer (PB) [pH 7.0,total phosphate concentration 0.07M]. After filtering and acidifyingwith phosphoric acid, triasulfurone is re-extracted 3 times with 75 mlof CH₂ Cl₂. After evaporating off the solvent, the sample is dissolvedin 10 ml of PB buffer and purified by means of filtration.

Method (B): The extraction according to method (A) is repeated with twofurther samples and the last organic phase is purified further byshaking with an aqueous hydrogen carbonate solution (5%) [Iwanzik andEgli (1989)]. After the addition of tetrabutylammonium hydrogencarbonate, triasulfurone is re-extracted in dichloromethane/n-hexane(80:20). After evaporating off the organic phase, triasulfurone is takenup in 10 ml of PB buffer. Before the sample is used in the ELISA test,it is diluted in PBS Tween 0.1% in a ratio of 1:20 or 1:40.

Method (C): In this case the extraction by shaking is effected usingtetrabutylammonium hydroxide directly with the methanol/PB extract. Theaqueous phase is then transferred to a liquid-liquid partitioningcartridge [ClinElut® No. 1010, Analytichem International, Harbor City,Calif.] and washed with 30 ml of n-hexane. The triasulfurone is elutedwith dichloromethane/n-hexane (60:40). The organic phase is evaporatedand the residue that remains is taken up in PBS.

EXAMPLE 8.2 Analysis of Water Samples

For competitive ELISA, 100 μl of PBS Tween buffer in a tenfoldconcentration are added to 850 μl of a water sample. The batch is thenincubated with 50 μl of the anti-triasulfurone antibody.

II. Results

1 ) Preparation of monoclonal antibodies

(a) Starting from 5 fusion events using mice that have been immunisedwith the KLH conjugate prepared in accordance with Example 1.3.1[triasulfurone conjugate of formula (IV)], a hybridoma is obtained thatproduces a monoclonal antibody having a high degree of affinity towardstriasulfurone [MAb 4134-40-1 ]. The fusion efficiency is approximately82%.

(b) Starting from 5 fusion events using mice that have been immunisedwith the KLH conjugate prepared in Example 1.3.2 [triasulfuroneconjugate of formula (V)], 19 hybridomas are obtained that produce amonoclonal antibody having a very high degree of affinity towardstriasulfurone. On the basis of their cross-reactivity patterns, 2 groupsof monoclonal antibodies, represented by MAb 4147-19-4 and MAb 4149-1-1,can be distinguished. Both MAbs belong to the IgG 1 isotype.

The cross-reactivity patterns of those two MAbs are very different, asTable 1 shows. In contrast to MAb 4147-19-4, MAb 4149-1-1 exhibitspronounced cross-reactivity with the hydroxylated triasulfurones.

MAb 4147-19-4, on the other hand, exhibits cross-reactivities with anumber of other triasulfurone analogs, especially with cinosulfurone. Inthe latter case, the cross-reactivity is 150%.

The lower detection limit for triasulfurone using the MAbs according tothe invention (in buffer) is in the range of from 0.01 to 1 ng/ml ofbuffer. The corresponding I₅₀ value is 0.09 ng/ml for MAb 4147-19-4 and0.05 ng/ml for MAb 4149-1-1.

Analysis of Extracted Soil Samples

Triasulfurone is added in different concentrations to extracts from fivedifferent standard soil samples of known composition and then determinedby means of an ELISA test using MAb 4147-19-4 and MAb 4149-1-1. Theresults are shown in Tables 2, 3 and 4.

The results show that the MAbs according to the invention areoutstandingly suitable for the detection of triasulfurone in soilsamples using an ELISA assay. The lower detection limit is approximately0.1 ppb.

II. DEPOSIT

The hybridoma cell lines prepared and used within the context of thepresent invention have been deposited under deposit numbers ECACC 90021702, ECACC 9002 1703 and ECACC 9002 1704 with the `European Collectionof Animal Cell Cultures` (ECACC) in Salisbury, UK, a recognisedinternational depository, in accordance with the requirements of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. A declaration ofthe viability of the deposited samples is issued by the saidinternational depository.

    ______________________________________                                                                         Date of                                                  Date of     Deposit  certificate                                  Cell line   deposit     number   of viability                                 ______________________________________                                        hybridoma   17.02.1990  9002 1702                                                                              17.02.1990                                   clone 4134-40-1                                                               hybridoma   17.02.1990  9002 1703                                                                              17.02.1990                                   clone 4147-19-4                                                               hybridoma   17.02.1990  9002 1704                                                                              17.02.1990                                   clone 4149-1-1                                                                ______________________________________                                    

III. MEDIA AND BUFFERS

    ______________________________________                                        (A)  RPMI 1640 medium                                                              RPMI 1640 (Seromed) with the following                                        additives:                                                                    calf serum                 15%                                                L-glutamine              4       mM                                           gentamycin               0.01%                                                sodium pyruvate          1       mM                                           2-mercaptoethanol        50      μM                                        insulin                  5       μM                                        transferrin              5       μM                                        selenium (ITS)           5       μM                                   (B)  HAT medium                                                                    1 liter of RPMI 1640 medium with 20 ml                                        addition of HAT conc. (50×) from                                        Boehringer, having the following                                              composition:                                                                  hypoxanthine 680         5.0     mg/l                                         amino                    8.8     mg/l                                         thymidine                193.8   mg/l                                    (C)  HT medium                                                                     1 liter of RPMI 1640 medium with 20 ml                                        addition of HT conc. (50×) from                                         Boehringer, having the following                                              composition:                                                                  hypoxanthine 680         5.0     mg/l                                         thymidine                193.8   mg/l                                    (D)  BSS buffer [Earle's salt solution, without Ca                                 and Mg, pH 7.4]                                                               KCl                      7.3     mM                                           NaCl                     116.0   mM                                           NaHCO.sub.3              26.0    mM                                           NaH.sub.2 PO.sub.4.2H.sub.2 O                                                                          1.0     mM                                           glucose                  5.5     mM                                           phenol red               48.0    μM                                        1% (v/v) addition of a penicillin/                                            streptomycin solution (Seromed) [10,000 U                                     penicillin, 10 mg/ml streptomycin]                                       (E)  Sodium carbonate buffer [pH 9.6]                                              Na.sub.2 CO.sub.3        477.0   mg                                           NaHCO.sub.3              879.0   mg                                           NaN.sub.3                1.8     mg                                           ad 300 ml H.sub.2 O                                                      (F)  PBS buffer [pH 7.0]                                                           NaCl                     8.5     g                                            Na.sub.2 HPO.sub.4.2H.sub.2 O                                                                          1.28    g                                            NaH.sub.2 PO.sub.4.2H.sub.2 O                                                                          0.436   g                                            ad 1000 ml H.sub.2 O                                                     (G)  PBS TWEEN 20 [0.1%]                                                           1 ml Tween 20 (Serva) + 1000 ml PBS                                      (H)  PBS BSA [1%]                                                                  BSA                      5.0     g                                            NaN.sub.3 (0.5M)         3.0     ml                                           ad 500 ml PBS                                                            (I)  Substrate buffer [diethanolamine buffer,                                      pH 9.8]                                                                       diethanolamine           97.0    ml                                           NaN.sub.3 (0.5M)         6.0     ml                                           MgCl.sub.2.6H.sub.2 O    100.0   mg                                           ad 1000 ml H.sub.2 O, adjustment of the pH to 9.8                             with conc. HCl                                                           ______________________________________                                    

Preparation of the substrate: immediately before use a substrate tablet(=5 mg) of the p-nitrophenyl phosphate substrate (Sigma 104) isdissolved in 5 ml of substrate buffer.

IV. BIBLIOGRAPHY

Ahmad I and Crawford G, J. Agric. Food Chem., 38: 138-141, 1990

Campbell AM, "Monoclonal Antibody Technology", in: Laboratory Techniquesin Biochemistry and Molecular Biology; Burdon, RH; Knippenberg PH(Eds.); Elsevier: Amsterdam, 1984; Vol.13, pp. 120-184

DeLuca, "Immunofluorescence Analysis", in: Antibody As a Tool,Marchalonis et al, John Wiley & Sons, Ltd., pp 189-231 (1982)

Ercegovich CD et al, J. Agric. Food Chem., 29: 559-563, 1981

Feng et al, J. Agric. Food Chem., 38: 159-163, 1990

Fleeker J, J. Assoc. Off. Anal. Chem., 70: 874-878, 1987

Hargrave HS and Merkle MG, Weed Sci., 19; 1971

Iwanzik W et al, Z. PflKrankh. PflSchutz, Suppl. XI: 301-310, 1988

Kawamura H, Berzojsky JA, J. Immunol., 136: 58, 1986

Kelley M et al, J. Agric. Food Chem., 33: 962-965, 1985

Kohler G, Milstein, Nature, 256: 495-497, 1975

Kulkarni NP et al, Cancer Res., 41: 2700-2706, 1981

Littlefield JW, Science, 145: 709, 1964

Newsome WH, J. Agric. Food Chem., 33: 528-530, 1985

Raab GM, Clin. Chem., 29: 1757-1761, 1983

Schlaeppi J-M et al, J. Agric. Food Chem., 37: 1532-1538, 1989

Shulman M et al, Nature, 276: 269-270, 1978

Stocker JW et al, Hoffmann-La Roche Research Disclosure, 21713: 155-157,1982

van Rensburg E, Analyst. 110: 733., 1985

Wie SI, Hammock BD, J. Agric. Food Chem., 30: 949-957, 1982

Zahnow EW, J. Agric. Food Chem., 30: 854-857, 1982

Patent Literature

U.S. Pat. No. 4,530,786

EP-A 0 044 808

V. TABLES

                  TABLE 1                                                         ______________________________________                                        Cross-reactivities of various triasulfurone analogs with                      MAb 4134-40-1, MAb 4147-19-4 and MAb 4149-1-1                                        MAb 4134-40-1                                                                           MAb 4147-19-4                                                                             MAb 4149-1-1                                              (a)     (b)     (a)   (b)   (a)   (b)                                         I       Cross-  I     Cross-                                                                              I     Cross-                                      50      reac-   50    reac- 50    reac-                                       (ng/    tivity  (ng/  tivity                                                                              (ng/  tivity                             Compound ml)     (%)     ml)   (%)   ml)   (%)                                ______________________________________                                        triasulfurone                                                                          7.2     100.0    0.09 100.0 0.05  100.0                              A          550   1.3     21.4  0.4   0.16  31.3                               B        >1000   <0.7      85  0.1   0.04  125.0                              C        >1000   <0.7    >1000 <0.01  1000 <0.01                              D        >1000   <0.7    >1000 <0.01 >1000 <0.01                              E        >1000   <0.7    >1000 <0.01 >1000 <0.01                              F        >1000   <0.7    >1000 <0.01 >1000 <0.01                              G        >1000   <0.7     0.06 150.0 2.3   2.2                                H          30    24.0     0.13 69.2  16.2  0.3                                I        >1000   <0.7    >1000 <0.01 >1000 <0.01                              J        >1000   <0.7    >1000 <0.01 >1000 <0.01                              K        >1000   <0.7    >1000 <0.01 >1000 <0.01                              L        >1000   <0.7    14.2  0.6   >1000 <0.01                              M        >1000   <0.7    14.3  0.6     420  0.01                              N        >1000   <0.7    >1000 <0.01 >1000 <0.01                              O        >1000   <0.7    81.5  0.1     600 <0.01                              P        >1000   <0.7    >1000 <0.01 >1000 <0.01                              Q        >1000   <0.7    >1000 <0.01 >1000 <0.01                              R        2.1     342.9   1.9   4.7   0.02  250.00                             S        6.0     120.0   2.6   3.5   0.02  250.00                             T        0.9     800.0   17.6  0.5   0.01  500.00                             ______________________________________                                         (a) Inhibitor concentration that reduces the ELISA signal by 50% in           comparison to the control.                                                    (b) (triasulfurone conc. for 50% inhibition/concentration of the              triasulfurone analog for 50% inhibition) × 100.                    

The triasulfurone analogs A to T are shown below with the aid of theirstructural formulae.

    __________________________________________________________________________     ##STR5##                               (I)                                   __________________________________________________________________________     ##STR6##                                                                              R.sub.4   R.sub.2                                                                           R.sub.5                                                                            R.sub.6                                                                            X   Y                                        __________________________________________________________________________    (A)      OCH.sub.2 CH.sub.2 Cl                                                                   H   OCH.sub.3                                                                          CH.sub.3                                                                           OH  H                                        (B)      OCH.sub.2 CH.sub.2 Cl                                                                   H   OCH.sub.3                                                                          CH.sub.3                                                                           H   OH                                       (O) methsulfur-                                                                        COOCH.sub.3                                                                             H   OCH.sub.3                                                                          CH.sub.3                                                                           H   H                                        one-methyl                                                                    (M) chloro-                                                                            Cl        H   OCH.sub.3                                                                          CH.sub.3                                                                           H   H                                        sulfurone                                                                     (K) tribenurone-                                                                       COOCH.sub.3                                                                             CH.sub.3                                                                          OCH.sub.3                                                                          CH.sub.3                                                                           H   H                                        methyl                                                                        reiauleuone                                                                            OCH.sub.2 CH.sub.2 Cl                                                                   H   OCH.sub.3                                                                          CH.sub.3                                                                           H   H                                        (G) cinosulfurone                                                                      OCH.sub.2 CH.sub.2 OCH.sub.3                                                            H   OCH.sub.3                                                                          OCH.sub.3                                                                          H   H                                        (H)      SCH.sub.2 CH.sub.2 F                                                                    H   OCH.sub.3                                                                          CH.sub.3                                                                           H   H                                         ##STR7##                                                                     (J) sulfometurone-                                                                     COOCH.sub.3                                                                             H   CH.sub.3                                                                            CH.sub.3                                         methyl                                                                        (I) primisulfurone                                                                     COOCH.sub.3                                                                             H   OCHF.sub.2                                                                          OCHF.sub.2                                        ##STR8##                                                                     (L) thiameturone-                                                                      COOCH.sub.3                                                                             H   OCH.sub.3                                                                          CH.sub.3                                          methyl                                                                         ##STR9##                                                                     (N) bensulfurone-                                                                      COOCH.sub.3                                                                             H   OCH.sub.3                                                                          OCH.sub.3                                         methyl                                                                         ##STR10##                                                                    (P) nicosulfurone-                                                                     CON(CH.sub.3).sub.2                                                                     H   OCH.sub.3                                                                          OCH.sub.3                                         methyl                                                                        (Q) DPX 9636                                                                           SO.sub.2 CH.sub.2 CH.sub.3                                                              H   OCH.sub.3                                                                          OCH.sub.3                                         __________________________________________________________________________             R.sub.7                                                                              R.sub.8                                                                              R.sub.9                                                                              R.sub.10                                         ##STR11##                                                                    (C)      H      H      Cl    H                                                (D)      H      H      OH    H                                                (E)      H      OH     Cl    H                                                (S)      H      H      Cl    CONH.sub.2                                       (R)      H      H      Cl    COOCH.sub.2 CH.sub.2 CH.sub.3                    (T)      H      H      Cl    CON(CH.sub.2 CH.sub.3).sub.2                     (F)                                                                                     ##STR12##                                                           __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Percentage of triasulfurone found in extracts of standard soil samples        after addition of triasulfurone to those extracts.                                                   MAb 4147-19-4      MAb 4149-1-1                                               tria-                                                                              triasulfurone                                                                        triasulfurone                                                                        triasulfurone                                                                        triasulfurone                        Soil constituents                                                                            sulfurone                                                                          (b)    (c)    (b)    (c)                                  humus                                                                             sand                                                                             silt                                                                             clay added                                                                              measured                                                                             found  measured                                                                             found                        Soil sample                                                                           (%) (%)                                                                              (%)                                                                              (%)                                                                              pH                                                                              ppb  ppb    (%)    ppb    (%)                          __________________________________________________________________________    Vetroz  9.3 18.1                                                                             60.4                                                                             21.5                                                                             7.3                                                                             0    0.17          0.17                                (Switzerland)          0.1  0.26   90     0.23   60                                                  0.3  0.44   90     0.47   100                                                 1    1.11   94     1.10   93                           Stein   5.0 43.0                                                                             17.4                                                                             34.6                                                                             7.1                                                                             0    0.06          0.07                                (Switzerland)          0.1  0.13   70     0.20   130                                                 0.3  0.39   110    0.39   107                                                 1    1.14   108    1.04   97                           Collombey                                                                             1.4 83.9                                                                             13.6                                                                             2.5                                                                              7.4                                                                             0    0.09          0.06                                (Switzerland)          1    1.08   99     1.04   98                           Les Evouettes                                                                         2.6 25.7                                                                             64.0                                                                             10.3                                                                             6.2                                                                             0    0.10          0.11                                (Switzerland)          1    0.91   81     1.00   89                           Speyer  1.0 93.0                                                                             3.1                                                                              2.9                                                                              7.4                                                                             0    0.04          0.07                                (Germany)              10   1.03   99     0.95   88                           __________________________________________________________________________     (a) Soil extracts prepared in accordance with Method A [see Example 8.1]-     (b) Calculated on the basis of standards of triasulfurone produced in PBS     Tween (average of 5 determinations)                                           (c) [ppb measured after addition - ppb before addition)/ppb added] .times     100.                                                                     

                  TABLE 3                                                         ______________________________________                                        Soil-matrix effect: Comparison of different extraction methods.                                    MAb 4147-19-4                                                                             MAb 4149-1-1                                                      triasulfurone                                                                             triasulfurone                                          (a)        (b)         (b)                                                    extraction measured    measured                                     Soil sample                                                                             method     ppb         ppb                                          ______________________________________                                        Vetroz    A          0.17        0.17                                                   B          0.03        0.02                                                   C (Expt. 1)                                                                              0.02        nd                                                        (Expt. 2)                                                                             0.07        0.16                                         Les Evouettes                                                                           A          0.10        0.11                                                   B          0.02        0.01                                                   C (Expt. 1)                                                                              0.06        nd                                                        (Expt. 2)                                                                             0.07        0.19                                         Stein     A          0.06        0.07                                                   C          0.01        0.09                                         Collombey A          0.09        0.06                                                   C          0.03        0.10                                         Speyer    A          0.04        0.07                                                   C          0.02        0.12                                         ______________________________________                                         (a) see Example 8.1                                                           (b) calculated on the basis of triasulfurone standards produced in PBS        Tween (average of 4 to 8 determinations per experiment)                       nd: not determined                                                       

                  TABLE 4                                                         ______________________________________                                        Recovery of triasulfurone from various                                        triasulfurone-enriched soil samples (a)                                                       triasulfurone found                                                           (MAb 4147-19-4)                                                       triasulfurone                                                                           (b)    (c)     (d)                                          Soil sample                                                                             added, ppb  ppb    (%)   SD    (CV)                                 ______________________________________                                        Vetroz    0.1         0.14   (120) 0.023 (16.4)                               (Switzerland)                                                                           0.5         0.34   (64)  0.033  (9.7)                                         1           0.68   (66)  0.115 (16.9)                                         10          6.60   (66)  0.801 (12.1)                               (Expt. 2) 0.1         0.19   (120) 0.021 (11.1)                                         0.3         0.31   (80)  0.017  (5.5)                                         1           0.60   (53)  0.046  (7.7)                               Les Evouettes                                                                           0.5         0.37   (62)  0.074 (20.0)                               (Switzerland)                                                                           1           0.71   (65)  0.184 (25.9)                                         10          7.41   (74)  1.129 (15.2)                               Stein     0.1         0.12   (110) 0.023 (19.2)                               (Switzerland)                                                                           0.3         0.25   (80)  0.008  (3.2)                                         1           0.63   (62)  0.040  (6.4)                               ______________________________________                                         (a) Soil samples extracted in accordance with Method C [see Example 8.1].     (b) Calculated on the basis of a standard produced in PBS Tween (average      of 4 determinations)                                                          (c) [(ppb measured after addition - ppb before addition)/ppb added]           × 100                                                                   (d) SD, standard deviation; CV, coefficient of variation                 

What is claimed is:
 1. A method for the preparation of a monoclonalantibody having a high degree of specificity and affinity towards one ormore sulfonylurea herbicides, wherein(a) a suitable sulfonylurea linkingcomponent comprising the sulfonamide moiety of the target moleculeobtainable by hydrolytical cleavage of the sulfonylurea compound at thesulfonamide function and a spacer fragment is conjugated with a suitablehigh molecular weight carrier molecule; (b) a donor animal is immunisedwith the conjugate prepared in accordance with (a); (c) immunocompetentB cells are isolated from the immunised donor animal; (d) the saidimmunocompetent B cells are fused with tumor cells capable of continuouscell division; (e) the resulting fusion product is isolated and, afterselection, the hybridoma cells that produce the desired antibody arecloned and (f) the said hybridoma cells are cultured in vitro or in vivoin order to produce monoclonal antibodies, wherein said sulfonylurealinking component is the stabilized fragment (B), ##STR13## wherein R'is COOH, NH₂ or SH and n is an integer from 1 to
 10. 2. A methodaccording to claim 1 wherein R' in formula (B) is COOH and n is aninteger from 1 to
 6. 3. A method according to claim 2 wherein R' is COOHand n=2.
 4. A hybridoma cell line that has the distinguishingcharacteristics of ECACC 9002 1702, and the clones and subclones thereofincluding mutants and variants that still have the distinguishingcharacteristics of the starting material.
 5. A hybridoma cell line thathas the distinguishing characteristics of ECACC 9002 1703, and theclones and subclones thereof including mutants and variants that stillhave the distinguishing characteristics of the starting material.
 6. Ahybridoma cell line that has the distinguishing characteristics of ECACC9002 1704, and the clones and subclones thereof including mutants andvariants that still have the distinguishing characteristics of thestarting material.
 7. A monoclonal antibody or a derivative thereofobtainable from a hybridoma cell line that has the distinguishingcharacteristics of ECACC 9002 1702 or from a mutant or variant thereof.8. A monoclonal antibody or a derivative thereof obtainable from ahybridoma cell line that has the distinguishing characteristics of ECACC9002 1703 or from a mutant or variant thereof.
 9. A monoclonal antibodyor a derivative thereof obtainable from a hybridoma cell line that hasthe distinguishing characteristics of ECACC 9002 1704 or from a mutantor variant thereof.
 10. A composition for the immunological detection oftriasulfurone in the form of a ready-to-use test kit, the said test kitcomprising, in addition to the carrier materials, reagents and otheradditives customarily used, at least one monoclonal antibody that isobtainable from a hybridoma cell line having the distinguishingcharacteristics of ECACC 9002 1702, or from clones or subclones thereof.11. A composition for the immunological detection of triasulfurone inthe form of a ready-to-use test kit, the said test kit comprising, inaddition to the carrier materials, reagents and other additivescustomarily used, at least one monoclonal antibody that is obtainablefrom a hybridoma cell line having the distinguishing characteristics ofECACC 9002 1703, or from clones or subclones thereof.
 12. A compositionfor the immunological detection of triasulfurone in the form of aready-to-use test kit, the said test kit comprising, in addition to thecarrier materials, reagents and other additives customarily used, atleast one monoclonal antibody that is obtainable from a hybridoma cellline having the distinguishing characteristics of ECACC 9002 1704, orfrom clones or subclones thereof.
 13. A method for the immunologicaldetection of triasulfurone in a sample comprising contacting said samplewith a monoclonal antibody obtainable from a hybridoma cell line thathas the distinguishing characteristics of ECACC 9002 1702, or fromclones or subclones thereof, and detecting the antibody-triasulfuronecomplex in a standard immunoassay.
 14. A method for the immunologicaldetection of triasulfurone in a sample comprising contacting said samplewith a monoclonal antibody obtainable from a hybridoma cell line thathas the distinguishing characteristics of ECACC 9002 1703, or fromclones or subclones thereof, and detecting the antibody-triasulfuronecomplex in a standard immunoassay.
 15. A method for the immunologicaldetection of triasulfurone in a sample comprising contacting said samplewith a monoclonal antibody obtainable from a hybridoma cell line thathas the distinguishing characteristics of ECACC 9002 1704, or fromclones or subclones thereof, and detecting the antibody-triasulfuronecomplex in a standard immunoassay.